Aortic valve disease is treated by replacing the diseased valve with either a mechanical or an animal tissue-based artificial heart valve. Unfortunately, neither device can provide good quality of life. Tissue engineering technologies offer the promise of a limitless supply of living tissues, such as replacement heart valves. The conventional approach to tissue engineering involves seeding cells on a biodegradable matrix, implanting it into the patient, and expecting a new valve to regrow as the matrix slowly degrades. Thus far, this approach has not been very successful because cardiovascular tissues are complex and have a low capacity for self-repair. We have been working on an alternative approach that we think is more appropriate for the aortic valve - the fabrication of the entire leaflet microstructure in vitro from the appropriate matrix molecules. We have been able to fabricate (i) collagen fiber bundles, both straight and branched, (ii) elastin tubes and sheets, and (iii) a viscoelastic glycosaminoglycan (GAG) matrix. Such an approach to tissue engineering (i) does not require regrowth of morphologically complex tissues, (ii) provides a matrix that can withstand cardiac loads immediately upon implantation, and (iii) can be designed and fabricated using conventional engineering principles. The GAG matrix is based on divinylsulfone-crosslinked hyaluronan (hylan), the collagen fiber bundles are fabricated using directed collagen gel shrinkage, and the elastin sheets and tubes have been grown on both the hylan and the collagen fiber bundles. Our next steps are to (i) improve the fabrication process of each of these components, (ii) improve their mechanical properties, and (iii) assemble the components to produce an aortic valve cusp with the appropriate mechanical properties. To this end we will (i) make use of dynamic cell culture to increase matrix synthesis and improve the mechanical properties of our constructs, and (ii) texturize hylan gels using UV and gamma irradiation to improve cell penetration and matrix adhesion. Once the material properties of the leaflet components are improved, they will be assembled into a composite aortic valve cusp and evaluated mechanically. Through this project, we aim to demonstrate that a tissue engineered valve cusp can be fabricated by manipulating biologic molecules in vitro using conventional biochemical and cell culture methodologies.